Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are central to sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation (RdDM) pathway. Here, we determined structure of a DCL3-pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated-A1 of the guide strand and the 1-nt 3′-overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated-end of the guide RNA and dual cleavage sites formed by the paired RNaseIII domains. These structural studies, complemented by functional data, reveal insights into the dicing principle for Dicers in general. This dataset includes small RNA-seq to understand the in vivo molecular mechanism of Arabidopsis DCL3 protein.

在真核生物中，小RNA（small RNAs，sRNAs）在多种生物学过程中发挥关键作用。Dicer核酸内切酶（Dicer endonucleases）是小RNA生物发生过程的核心因子。在植物中，DICER样蛋白3（DICER-LIKE PROTEIN 3，DCL3）可生成24核苷酸长度的小干扰RNA（small interfering RNAs，siRNAs），这些小干扰RNA决定了RNA指导的DNA甲基化（RNA-directed DNA methylation，RdDM）通路的特异性。本研究解析了处于具备切割活性状态的DCL3-前体siRNA复合物的结构。向导链的5′端磷酸化A1位点与互补链的1个核苷酸3′突出端，分别通过带正电的口袋与芳香族帽结构实现特异性识别。24核苷酸长度的siRNA的长度依赖性，源于向导RNA的5′磷酸化末端与成对RNase III结构域形成的双重切割位点之间的间距。这些结构研究结合功能实验数据，揭示了Dicer家族酶通用的切割机制。本数据集包含小RNA测序（small RNA-seq）数据，用于探究拟南芥DCL3蛋白的体内分子机制。